1. Field of the Invention
The present invention relates to a method for the immunological determination of a hemoglobin derivative, preferably glycated hemoglobin as a hemoglobin derivative, and especially hemoglobin A.sub.1c. The present invention also relates to a treating reagent to be used in the method of determination mentioned above.
2. Description of the Prior Art
Hemoglobin (Hb) is a respiratory pigment present in erythrocyte, which is largely responsible for oxygen transport. A hemoglobin molecule comprises four polypeptide subunits (respectively two a chain systems and .beta. chain systems), each subunit is formed by association of one globin protein and one heme molecule which is an iron-protoporphyrin complex. Major class of hemoglobin (more than 90%) found in normal adult hemoglobin (HbA: also referred to HbA.sub.0 for distinguishing from glycated hemoglobin HbA.sub.1 described hereinafter) having .alpha..sub.2 .beta..sub.2 subunits composition; and trace components such as HbA.sub.2 (.alpha..sub.2 .delta..sub.2) are also found in normal adult.
There are several classes of hemoglobin derivatives in the adult hemoglobin HbAs. The determination of the contents of such hemoglobin derivatives in blood has been gaining in importance under various medical conditions. Among classes of hemoglobin derivatives, glycated hemoglobin proves to be particularly important. This glycated hemoglobin is the generic term for designating the fractions, HbA.sub.1a1, HbA.sub.1a2, HbA.sub.1b, and HbA.sub.1c, into which HbA is classified by an ion-exchange resin fractionation. It is referred to as HbA.sub.1 (also designated as glycosylated hemoglobin). All of these subclasses of the glycated hemoglobins have the same primary structure, which is stabilized by formation of an aldimine (Schiff base) by the amino group of the N terminal valine in the .beta. subunit chain of normal hemoglobin HbA and glucose (or glucose-6-phosphate or fructose) followed by formation of a ketoamine by Amadori rearrangement.
Particularly, the glycated hemoglobin bound with glucose are called as HbA.sub.1c (glycosylated hemoglobin, which will be occasionally referred to as hemoglobin A.sub.1c hereinafter) and they constitute the greater part of glycated hemoglobins. The content ratio of glycosylated hemoglobin is proportional to the blood glucose level and ranges about 5% in the total hemoglobin Hb of normal human adult. It possibly rise to levels in the range of 8 to 16% in patients of diabetes. The determination of the amount of glycosylated hemoglobin HbA.sub.1c, therefore, is regarded as a good index for carbohydrate metabolism control. Further, since the ketoamine formed by a non-enzymatic reaction with blood glucose is stable, the glycosylated hemoglobins HbA.sub.1c will not decompose during the life of erythrocyte (an average of 120 days). The hemoglobin A.sub.1c content in blood, therefore, is construed as recording the blood glucose level in the past one to two months. As a result, the blood glucose level in the last two months or so can be estimated on the basis of the ratio of HbA.sub.1c to the total hemoglobin Hb. Thus, the analysis of the hemoglobin A.sub.1c in blood is utilized as an index which permits a long-term control of blood glucose level, unlike the short-term index such as blood glucose level which generally rises briefly after a meal.
Various methods have been developed for the analysis for the hemoglobin A.sub.1c. These methods generally are based on such techniques as electrophoresis, ion-exchange chromatography, and affinity chromatography. These methods invariably do not fit the clinical test which handles numerous samples because they require expensive analytical apparatuses and consume much time for analysis. In this respect, such immunological methods of analysis as the immunoassay which make use of antibodies to hemoglobin A.sub.1c are at an advantage in adopting relatively simple procedures and obviating use of much time. Particularly, since specific antibodies to the glycosylated N-terminal residue of HbA.sub.1c as specific antibodies to HbA.sub.1c are disclosed, for examples, in Unexamined Japanese Patent Publication (KOKAI) Nos. 8743/1989, 172064/1986, and 280571/1986, various immunological methods of determination of HbA.sub.1c have been developed (for example, Unexamined Japanese Patent Publication (KOKAI) Nos. 277967/1988 and 46566/1991).
In the field of the immunological determination of HbA.sub.1c, various treatments are attempted to realize analysis of greater sensitivity by treating the HbA.sub.1c in a given sample. Most of the methods adopted for such treatment, however, are not easily applied for an enzyme immunoassay, because the treatments in these method are mainly due to the denaturation process of HbA.sub.1c which is a glycoprotein.
Unexamined Japanese Patent Publication No. 155268/1989 (corresponding to U.S. Pat. No. 4,970,171 and EP-A-0315864), for example, discloses a method for determining HbA.sub.1c in blood by denaturing hemoglobin in blood with thiocyanate and further converting it to methhemoglobin with an oxidizing agent such as ferricyanide. In this method, hemoglobin is converted or denatured to methhemoglobin having a specific absorption peak at 540 nm, which can be easily detected to facilitate the determination of the total hemoglobin content. The oxidizing agent enhances the efficiency of denaturation of the thiocyanate denaturing agent, thereby the sensitivity of detection of HbA.sub.1c in the sample is improved. Although it is desirous that this treatment can be applied to an enzyme immunoassay for analysis of the HbA.sub.1c, it is difficult to realize such an application, because the labelled enzyme is also affected and inactivated by thiocyanate or the oxidizing agent. Although the enzyme immunoassay may be effectively adopted on the condition that the thiocyanate and the oxidizing agent are removed from the sample after the completion of the treatment, the cumbersome operations are needed for the removal of the agents. Further, since this method uses a cyanide harmful to the environment, the waste liquid problem is raised.
Unexamined Japanese Patent Publication (KOKAI) No. 20452/1989 (corresponding to U.S. Pat. No. 4,800,167 and EP-A-0286915) discloses a method for the determination of total hemoglobin content without the use of a cyanide. This method uses as a denaturing reagent an aqueous solution of polyvinyl pyrrolidone (PVP) which is alkalinized to a level in the range of pH 12 to 14. This denaturing reagent is intended to use the PVP for stabilizing the hemoglobin, which is solubilized with a high alkali, and give rise to a product having the main absorption peak at the wavelength of about 575 nm. The application of this method for the determination of HbAic is conceivable. However, such application it proves unfavorable because the sugar moiety of the HHbA.sub.1c molecule is possibly cleavaged or broken in the presence of a strong alkali to change the antigenicity of the HbA.sub.1c molecule. The determination of HbA.sub.1c by the enzyme immunoassay has also a disadvantage in suffering the enzymatic activity of the labelling enzyme of the antibody to be suppressed since most enzymes have their optimum pH values generally in the neutral range (pH 6 to 8). More importantly, the immunological reaction (antigen-antibody binding reaction) itself is possibly suppressed by a high pH condition. Therefore, this treatment method is not easily applied to the immunoassay.
Unexamined Japanese Patent Publication (KOKAI) No. 11510/1996 (corresponding to DE 4206932A) discloses a method wherein a blood specimen is treated with a hemolysis reagent containing an ionic detergent (surfactant) having a pH value in the range of 5.0 to 9.5. The total hemoglobin content in the hemolyzed sample is analyzed by calorimetric determination. The amount of the hemoglobin A.sub.1c in the hemolyzed sample is analyzed by the immunological determination. Examples of ionic detergents to be used in this treatment include anionic detergents such as SDS (sodium dodecyl sulfate) and cationic detergents such as TTAB (tetradecyl trimethyl ammonium bromide). These ionic detergents have been well known as hemolyzing reagents in the art. Since these ionic detergents also have an action of denaturing proteins, they have an adverse effect on the labelled enzyme and inevitably inhibit the enzymatic activity thereof. In the heterogeneous enzyme immunoassay which necessitates B/F separation, the ionic detergent can be removed simultaneously with the B/F separation after the antigen-antibody binding reaction, so that the enzymatic activity may be prevented from the adverse effect by the ionic detergent. When the treatment method with ionic detergents adopts to the homogeneous enzyme immunoassay which does not necessitate the B/F separation, however, the ionic detergent remains in the treated sample solution and inhibit the enzymatic activity of the labelling enzyme, resulting in less sensitivity for practical uses.
Japanese Patent Publication No. 23891/1995 (EP-A-185870, U.S. Pat. No. 4,647,654) discloses a method wherein a protein such as HbA.sub.1c is treated and denatured with a chaotropic reagent thereby exposing the epitope of the protein and enhancing the affinity thereof for an antibody. This method is unfit for quick determination because the step of the denaturation consumes a long time ranging from one to several hours at temperatures below 37.degree. C. Since the denaturing reagent used in this method is guanidine hydrochloric acid, urea, SDS, or protease, it is destined to inactivate the enzymatic activity in the same manner as in the prior techniques mentioned above. In the homogeneous enzyme immunoassay, therefore, this method is practically incapable of attaining necessary determination of HbA.sub.1c.